3D Imaging Reveals Complex Microvascular Remodeling in the Right Ventricle in Pulmonary Hypertension

Abstract

Background: Pathogenic concepts of right ventricular (RV) failure in pulmonary arterial hypertension focus on a critical loss of microvasculature. However, the methods underpinning prior studies did not take into account the 3-dimensional (3D) aspects of cardiac tissue, making accurate quantification difficult. We applied deep-tissue imaging to the pressure-overloaded RV to uncover the 3D properties of the microvascular network and determine whether deficient microvascular adaptation contributes to RV failure.

Methods: Heart sections measuring 250-µm-thick were obtained from mice after pulmonary artery banding (PAB) or debanding PAB surgery and properties of the RV microvascular network were assessed using 3D imaging and quantification. Human heart tissues harvested at the time of transplantation from pulmonary arterial hypertension cases were compared with tissues from control cases with normal RV function.

Results: Longitudinal 3D assessment of PAB mouse hearts uncovered complex microvascular remodeling characterized by tortuous, shorter, thicker, highly branched vessels, and overall preserved microvascular density. This remodeling process was reversible in debanding PAB mice in which the RV function recovers over time. The remodeled microvasculature tightly wrapped around the hypertrophied cardiomyocytes to maintain a stable contact surface to cardiomyocytes as an adaptation to RV pressure overload, even in end-stage RV failure. However, microvasculature-cardiomyocyte contact was impaired in areas with interstitial fibrosis where cardiomyocytes displayed signs of hypoxia. Similar to PAB animals, microvascular density in the RV was preserved in patients with end-stage pulmonary arterial hypertension, and microvascular architectural changes appeared to vary by etiology, with patients with pulmonary veno-occlusive disease displaying a lack of microvascular complexity with uniformly short segments.

Conclusions: 3D deep tissue imaging of the failing RV in PAB mice, pulmonary hypertension rats, and patients with pulmonary arterial hypertension reveals complex microvascular changes to preserve the microvascular density and maintain a stable microvascular-cardiomyocyte contact. Our studies provide a novel framework to understand microvascular adaptation in the pressure-overloaded RV that focuses on cell-cell interaction and goes beyond the concept of capillary rarefaction.

Keywords: fibrosis; heart failure; microvascular rarefaction; microvessels; pulmonary arterial hypertension.

Figure 1:. 3D Quantification of the RV microvascular and Fibrosis Volume in PAB mice

(A) 1^st row: Three-dimensional (3D) projection of 250 µm-thick sections of the mid-ventricle stained with isolectin B4 (IB4) obtained from sham, week 1, week 4, and week 7 post-PAB male animals. The yellow rectangles outline regions of the RV free wall selected for further imaging and quantification (see Figure 1 and 2). Scale bar: 1 mm. 2^nd row: Maximum intensity projection (MIP) of 250 µm-thick z-stacks of the RV free wall stained with IB4. 3^rd row: 3D reconstruction of the IB4- and WGA-stained structures (i.e., microvasculature and fibrotic tissue). Scale bar (rows 2–3): 100 µm. (B) Microvascular volume and (C) tissue volume within the imaged area, both of which showed significant increase at PAB week 4 (w4) and week 7 (w7) compared to Sham and PAB week 1 (w1). (D) Microvascular density (i.e., the ratio of microvasculature volume to the whole tissue volume within the imaged area). (E) The percentage of fibrotic tissue volume relative to the whole tissue volume within the imaged area. (B)-(E) Each dot represents quantification from each animal. Black dots: male, red dots: female. N=6 animals for male sham, male PAB w1, female sham, female PAB w4, and female PAB w7. N=7 animals for male PAB w7 and female PAB w1. N=8 animals for male PAB w4. Data are expressed as mean ± SD, analyzed by the Kruskal-Wallis test for selected comparisons.

Figure 2:. Remodeling of the Microvascular Network in the Pressure Overloaded RV

(A) Filament tracing of IB4-stained microvasculature color-coded with orientation angle (1^st row), length (2^nd row), and diameter (3^rd row) of each microvascular segment in the RV free wall. Scale bar: 100 µm. (B) Ratio of the number of segments running in the radial direction to the number running in the circumferential direction of the RV free wall. (C) Mean segment length. (D) Mean diameter of segments. (E) Number of the segments normalized with the tissue volume. (B)-(E) Each dot represents quantification from each animal. Black dots: male, red dots: female mice. N=6 animals for male sham, male PAB w1, female sham, female PAB w4, and female PAB w7. N=7 animals for male PAB w7 and female PAB w1. N=8 animals for male PAB w4. Data are expressed as mean ± SD, analyzed by the Kruskal-Wallis test for selected comparisons.

Figure 3.. Preservation of Microvascular-Cardiomyocyte Contact Area

(A) 1^st row: 3D projection of 250 µm-thick vibratome sections of the mid-ventricle from mTmG-Myh6Cre mice dosed with tamoxifen subjected to PAB. Cardiomyocytes are labeled sparsely with GFP and stained with anti-GFP antibody. Scale bar: 1 mm. 2^nd row: Single optical section of RV free wall. Scale bar: 100 µm. 3^rd row: Maximum intensity projection (MIP) of areas in yellow rectangles of the 2^nd row. Cardiomyocytes were randomly selected for imaging from the RV free wall for the sham and PAB week 1 animals, and from non-fibrotic areas of the RV free wall in PAB week 4 and week 7 animals. 4^th row: 3D reconstruction of single cardiomyocyte and surrounding microvasculature from the RV of sham and PAB week 1 animals and non-fibrotic areas of PAB week 4 and week 7 animals. Contact area is visualized as yellow surface on the cardiomyocytes. 5^th row: MIP of areas in red rectangles of the 2^nd row. Cardiomyocytes were randomly selected from fibrotic areas of the RV free wall in PAB week 4 and week 7 animals. 6^th row: 3D reconstruction of single cardiomyocyte and surrounding microvasculature in the fibrotic areas of PAB week 4 and week 7 animals. Contact area is visualized as yellow surface on the cardiomyocytes. Scale bar for 3–6^th row: 50 µm. (B) Contact area between the microvasculature and cardiomyocytes was significantly reduced only in the fibrotic areas. Each dot represents the mean of contact area/cardiomyocyte volume of each animal and 30–50 cardiomyocytes/animal were quantified. N=6 animals for male sham, male PAB w1, female sham, female PAB w4, and female PAB w7. N=7 animals for male PAB w7 and female PAB w1. N=8 animals for male PAB w4. Data are expressed as mean ± SD, analyzed by the Kruskal-Wallis test followed by Dunn’s multiple comparisons.

Figure 4:. Fibrosis and Cardiomyocyte Hypoxic Stress

(A) 3D projection of the IB4-stained microvasculature with overlayed 3D reconstruction of WGA-stained fibrotic tissue obtained from PAB week 7 animal. Scale bar: 100 µm. (B) Magnified image of the red rectangle area in A. Upper panel: 3D reconstruction of the microvasculature. Lower panel: 3D reconstructions of the microvasculature and fibrosis. Yellow arrows indicate microvasculature fully surrounded by interstitial fibrosis. Scale bar: 20 µm. (C) Transmission electron microscopy image of cardiomyocytes (CM), microvasculature, and fibrosis in the RV showing extracellular matrix (ECM) fully surrounding the microvasculature, thereby preventing direct contact between endothelial cells (EC) and cardiomyocytes. RBC: red blood cell, FB: fibroblast, Scale bar: 2 µm. (D) Formalin-fixed paraffin-embedded section of the heart from sham and PAB week 7 mice stained with WGA, 𝛽-MyHC, and DAPI. Scale bar: 500 µm. Lower panels are magnified views of the area in yellow rectangle from the upper panel. Scale bar: 100 µm. (E) Quantification of the number of 𝛽-MyHC positive cardiomyocytes adjacent to fibrosis (WGA-positive tissue). Each dot represents quantification from each animal. N=4 animals for each group. Data are expressed as mean ± SD, analyzed by the unpaired Mann-Whitney U-test. (F) Western blotting of RV tissue homogenates with anti-hypoxyprobe (pimonidazole) antibody showing increased binding of pimonidazole in the RV of PAB mice. Each lane represents tissue homogenates from each animal. N=3 animals for each group.

Figure 5.. Ongoing Endothelial Cell Proliferation in End-stage RV Failure

(A): Fresh frozen tissue section of sham, day 4, week 1, 4, and 7 post-PAB heart stained with endothelial cell marker ERG and DAPI along with EdU detection. 2^nd-5^th row: Magnified view of areas in yellow rectangle from the 1^st row. Scale bar: 1 mm for 1^st row, 100 µm for 2^nd-5^th row. (B) The fraction of proliferating endothelial cells (EdU+ERG+ cells) among the endothelial cells (ERG+). (C) The fraction of proliferating non-endothelial cells (EdU+ERG- cells) among the proliferating cells (EdU+). (D) The fraction of proliferating cells (EdU+) among all types of cells (DAPI). Quantifications were done from six to seven randomly selected areas of the RV. Each dot represents quantification from each animal. N=3 animals for each group. Data are expressed as mean ± SD, analyzed by the Kruskal-Wallis test followed by Dunn’s multiple comparisons.

Figure 6:. Deep-tissue Imaging of the RV from Patients with Different Etiologies of PAH

(A) 1^st row: Sections from the hearts from declined donor with normal RV function and PAH cases with various etiologies. The red rectangles outline regions excised for deep tissue imaging. Scale bar 1 cm. 2^nd row: Maximum intensity projection (MIP) of the 250 µm-thick z-stacks of RV free wall stained with Ulex Europaeus Agglutinin I (UEA-I). The yellow rectangles outline regions of the RV free wall selected for further imaging and quantification (see Figure 6 and 7). Scale bar: 1 mm. 3^rd row: Representative single optical section (2D) of the RV free wall stained with UEA-I. 4^th row: MIP of the z-stacks of the area within the yellow rectangle. The capillary density (microvascular volume / tissue volume) is shown below the images. 5^th row: Representative single optical plane (2D) of the RV free wall stained with UEA-I and WGA. 6^th row: 3D reconstruction of the UEA-I- and WGA-positive structures (i.e., microvasculature and fibrotic tissue, respectively) created by Imaris. The percentage of fibrotic tissue volume relative to the whole tissue volume is shown below the images. Scale bar of 3^rd-6^th panel: 100 µm.

Figure 7:. Architectural Remodeling of the RV Microvasculature in PAH patients

(A) Filament model of UEA-I-stained microvasculature color-coded with orientation angle (1^st row), mean length (2^nd row), and mean diameter (3^rd row) of the microvascular segments. Scale bar: 100 µm. (B) Histogram showing the distribution of the length of each microvascular segment. (C) Density plot showing the relationship between the length and diameter of each microvascular segment.